Robotic Mammosphere Assay for High-Throughput Screening in Triple-Negative Breast Cancer.

In order to identify novel treatment principles specifically affecting cancer stem cells in triple-negative breast cancer, we have developed a high-throughput screening method based on the mammosphere and anoikis resistance assays allowing us to screen compounds using a functional readout. The assay was validated against manual protocols and through the use of positive controls, such as the response to hypoxia and treatment with the known cancer stem cell-targeting compound salinomycin. Manual and robotic procedures were compared and produced similar results in cell handling, cell cultures, and counting techniques, with no statistically significant difference produced from either method. The variance between samples processed manually versus robotically was no greater than 0.012, while Levene's test of significance was 0.2, indicating no significant difference between mammosphere data produced manually or robotically. Through the screening of 989 FDA-approved drugs and a follow-up screen assessing the antineoplastic subgroup, we have identified three therapeutic compounds with the ability to modulate the breast cancer stem cell fraction in the triple-negative breast cancer cell line MDA-MB-231, highlighting their potential usage as stem cell-specific adjuvant treatments.

The unfolding and attempted refolding of the bacterial chaperone protein groEL (cpn60).

The unfolding of the bacterial chaperone protein groEL (cpn60) in solutions of guanidinium chloride (GdnHCl) has been studied. From the results of CD, fluorescence and light scattering, it is clear that major structural transitions in the protein occur over the range 1.0-1.5 M GdnHCl. The ATPase activity of the protein is lost at lower concentrations (0.75 M). After denaturation in concentrations of GdnHCl above 1.5 M, removal of the denaturing agent by dialysis results in very nearly complete regain of secondary structure (as judged by CD), but not the regain of correct tertiary or quaternary structure, or ATPase activity. The product was shown to be very sensitive to proteolysis by thermolysin, unlike the native protein, and not to show enhanced binding of ANS, a characteristic property of the 'molten globule' state of proteins. The results are discussed in relation to current information concerning the assembly of the groEL protein.

Conserved residues in the mechanism of the E. coli Class II FBP-aldolase.

The two classes of fructose-1,6-bisphosphate aldolase both catalyse the reversible cleavage of fructose 1,6-bisphosphate into dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. The Class I aldolases use Schiff base formation as part of their catalytic mechanism, whereas the Class II enzymes are zinc-containing metalloproteins. The mechanism of the Class II enzymes is less well understood than their Class I counterparts. We have combined sequence alignments of the Class II family of enzymes with examination of the crystal structure of the enzyme to highlight potentially important aspartate and asparagine residues in the enzyme mechanism. Asp109, Asp144, Asp288, Asp290, Asp329 and Asn286 were targeted for site-directed mutagenesis and the resulting proteins purified and characterised by steady-state kinetics using either a coupled assay system to study the overall cleavage reaction or using the hexacyanoferrate (III) oxidation of the enzyme bound intermediate carbanion to investigate partial reactions. The results showed only minor changes in the kinetic parameters for the Asp144, Asp288, Asp290 and Asp329 mutants, suggesting that these residues play only minor or indirect roles in catalysis. By contrast, mutation of Asp109 or Asn286 caused 3000-fold and 8000-fold decreases in the kcat of the reaction, respectively. Coupled with the kinetics measured for the partial reactions the results clearly demonstrate a role for Asn286 in catalysis and in binding the ketonic end of the substrate. Fourier transform infra-red spectroscopy of the wild-type and mutant enzymes has further delineated the role of Asp109 as being critically involved in the polarisation of the carbonyl group of glyceraldehyde 3-phosphate.

A functional role for a flexible loop containing Glu182 in the class II fructose-1,6-bisphosphate aldolase from Escherichia coli.

Class II fructose 1,6-bisphosphate aldolases (FBP-aldolases) catalyse the zinc-dependent, reversible aldol condensation of dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3-phosphate (G3P) to form fructose 1,6-bisphosphate (FBP). Analysis of the structure of the enzyme from Escherichia coli in complex with a transition state analogue (phosphoglycolohydroxamate, PGH) suggested that substrate binding caused a conformational change in the beta5-alpha7 loop of the enzyme and that this caused the relocation of two glutamate residues (Glu181 and Glu182) into the proximity of the active site. Site-directed mutagenesis of these two glutamate residues (E181A and E182A) along with another active site glutamate (Glu174) was carried out and the mutant enzymes characterised using steady-state kinetics. Mutation of Glu174 (E174A) resulted in an enzyme which was severely crippled in catalysis, in agreement with its position as a zinc ligand in the enzyme's structure. The E181A mutant showed the same properties as the wild-type enzyme indicating that the residue played no major role in substrate binding or enzyme catalysis. In contrast, mutation of Glu182 (E182A) demonstrated that Glu182 is important in the catalytic cycle of the enzyme. Furthermore, the measurement of deuterium kinetic isotope effects using [1(S)-(2)H]DHAP showed that, for the wild-type enzyme, proton abstraction was not the rate determining step, whereas in the case of the E182A mutant this step had become rate limiting, providing evidence for the role of Glu182 in abstraction of the C1 proton from DHAP in the condensation direction of the reaction. Glu182 lies in a loop of polypeptide which contains four glycine residues (Gly176, Gly179, Gly180 and Gly184) and a quadruple mutant (where each glycine was converted to alanine) showed that flexibility of this loop was important for the correct functioning of the enzyme, probably to change the microenvironment of Glu182 in order to perturb its pK(a) to a value suitable for its role in proton abstraction. These results highlight the need for further studies of the dynamics of the enzyme in order to fully understand the complexities of loop closure and catalysis in this enzyme.

Selection in complex genetic systems. I. The symmetric equilibria of the three-locus symmetric viability model.

The symmetric equilibria of the three-locus symmetric viability model are determined and their stability analyzed. For tight linkage there may be four stable equilibria, each characterized by having one pair of complementary chromosomes in high frequencies, with all others low. For looser linkage the only stable symmetric equilibrium is that with complete linkage equilibrium. For intermediate recombination values both types of equilibria may be stable. A new class of equilibria with all pairwise linkage disequilibria zero, but with third order linkage disequilibrium, has been discovered. It may be stable for tight linkage.

The reaction of GroEL (cpn 60) with the ATP analogue 2',3' dialdehyde ATP.

The reaction of the E. coli chaperonin GroEL (cpn 60) with the ATP analogue 2',3' oxidised ATP (oATP) has been studied. Treatment with the reagent leads to loss of the ATPase activity of GroEL in a pseudo-first-order fashion; this can be prevented by inclusion of ATP in the reaction mixture. Measurements of the stoichiometry of the reaction indicate that the loss of activity corresponds to the incorporation of about one oATP per subunit of GroEL. From analysis of the sequences of modified peptides it is proposed that the reaction probably occurs with one or both of the two cysteines Cys-457 and Cys-518, although the instability of the adduct(s) makes a definite identification of the site(s) of reaction difficult. The involvement of Cys side chains in the reaction with oATP was confirmed by using Nbs2 (5,5'-dithiobis(2-nitrobenzoate)) to estimate thiol groups in both modified and unmodified GroEL.

The response of rapidly formed adult human endothelial-cell monolayers to shear stress of flow: a comparison of fibronectin-coated Teflon and gelatin-impregnated Dacron grafts.

Endothelial cells labeled with indium 111-oxine were seeded in supraconfluent densities onto fibronectin-coated expanded polytetrafluoroethylene (ePTFE) and gelatin-impregnated Dacron graft segments. These grafts with rapidly formed endothelial-cell monolayers were then exposed to varying shear stresses at flow rates of 200 and 300 ml/min, using tissue culture medium in an artificial flow circuit. As the loss of radioactivity represented endothelial-cell loss, cell retention was calculated by the ratio of counts recorded at different time points during 2 hours of flow to initial counts. Although initial cell adherence to gelatin-impregnated Dacron graft segments was poor compared to ePTFE, once cells were attached they resisted shear stress of flow better at 200 ml/min and equally well at 300 ml/min. The cell retention on fibronectin-coated ePTFE was 55.4 +/- 12.9% at 200 ml/min and 56.5 +/- 15.2% at 300 ml/min; cell retention for gelatin-impregnated Dacron graft segments was 69.0 +/- 6.0% and 66.5 +/- 5.5%, respectively. Qualitatively scanning electron microscopy of both ePTFE and gelatin-impregnated Dacron graft segments showed patchy coverage of grafts with cells. There was preferential attachment of endothelial cells to the nodes on ePTFE, although on gelatin-impregnated Dacron graft segments, cells conformed to the Dacron fibers at different levels and directions with evidence of bridging in the gaps between individual fibers. This study shows conclusively that rapidly formed endothelial-cell monolayers on ePTFE and gelatin-impregnated Dacron graft segments resisted a shear stress of flow equal to that seen in a femoropopliteal vein graft with significant cell retention at 2 hours.

Adult human endothelial cell seeding using expanded polytetrafluoroethylene vascular grafts: a comparison of four substrates.

Prosthetic small-caliber vascular grafts give poorer patency rates than autogenous vein grafts, possibly because the former never spontaneously form endothelium. Animal studies have shown that endothelialization of prosthetic grafts can be encouraged by seeding endothelial cells into the graft at the time of surgery, resulting in improved patency. Information regarding the attachment characteristics of adult human endothelial cells to prosthetic grafts is, however, sparce. Laboratory experiments were performed by use of cell culture techniques to compare the attachment characteristics of adult human endothelial cells to expanded polytetrafluoroethylene graft material, both untreated and treated, with one of four protein substrates--preclotted blood, fibronectin, laminin, and type 4 collagen. Attachment characteristics were compared quantitatively by use of attachment assays and qualitatively by scanning electron microscopy. Attachment to untreated expanded polytetrafluoroethylene was poor but could be greatly improved by preclotting or precoating with any of the proteins, particularly fibronectin. In preclotted grafts seeded endothelial cells formed a virtually confluent monolayer after 1 hour. Cell attachment to the other grafts coated with protein was patchy and inconsistent. It is concluded that a rapid confluent endothelial lining within a prosthetic vascular graft is possible, and of the substrates examined, preclotted blood best encourages cell attachment to expanded polytetrafluoroethylene.

Metronidazole prophylaxis in colorectal surgery: the need for additional aminoglycoside?

A randomized double-blind trial was conducted in two phases to compare the efficacy of prophylactic oral and intravenous administration of kanamycin and metronidazole in preventing wound infection and other complications in patients undergoing colorectal surgery, and also to assess the need for inclusion of an aminoglycoside in the prophylactic regimen. Adequate prophylaxis did not depend on the route of antimicrobial administration, but wound infection rates were increased following exclusion of kanamycin.

The early diagnosis of acute gall-bladder disease: the accuracy of overnight eight-hour infusion cholangiography.

The reliability of overnight 8-hour infusion cholangiography in confirming the diagnosis of acute cholecystitis or biliary colic was assessed by a prospective study in 100 patients. 55 positive infusion cholangiograms were subsequently confirmed at operation in 45 patients and by cholecystogram in 10 patients who did not have surgical treatment. The absence of false positive examinations is of fundamental importance if early cholecystectomy is to be performed without the risk of an unnecessary laparotomy. Infusion cholangiography proved to be a safe, simple and reliable investigation in the confirmation of acute gall-bladder disease.

Spinal cord monitoring in patients with nonidiopathic spinal deformities using somatosensory evoked potentials.

Seventy-nine somatosensory evoked potentials were intraoperatively recorded in 52 patients undergoing spinal surgery for nonidiopathic spinal deformities. There were 37 true-negative, 28 true-positive (a significant change in the somatosensory evoked potential related to the surgical process), and 14 false-positive (a significant change in the somatosensory evoked potential not related to a surgical event) readings. There were, however, no postoperative neurologic deficits with any of the true-positive readings and no false negatives. Spinal and subcortical somatosensory evoked potentials gave few false-positive readings. True-positive somatosensory evoked potentials occurred in 44% of the patients with neuromuscular deformities, 17% with congenital deformities, 45% with Luque instrumentation, 22% with Harrington instrumentation, and none with fusion in situ. Fifty percent of the true positives occurred while the sublaminar wires were tightened. The predictive accuracy of intraoperative spinal cord monitoring in this patient population is not high, but the sensitivity to potentially harmful surgical events is high.

A clinical trial of Gingkco Biloba Extract in patients with intermittent claudication.

Thirty-seven patients with stage 2 peripheral vascular disease were randomised to receive a six month course of Gingkco Biloba Extract (Tanakan) or matching placebo. Assessment, by claudication distance, A/B ratio, Doppler ankle pressure response to exercise together with recovery time, and a 10 cm analogue scale (LAS) estimation of maximal pain severity, was performed before treatment, and at 6, 12 and 24 weeks. LAS scores were significantly improved after 24 weeks in patients receiving EgB, but not placebo. Claudication distance was significantly increased by Egb. Although claudication distance also increased in the placebo group, this was not significant. A/B ratio and Doppler ankle responses to exercise did not show any significant change in either group at any time interval, nor did the post exercise recovery time. Gingkco Biloba Extract is a safe and effective method of improving walking distance and reducing pain severity in patients with intermittent claudication, although Doppler studies have failed to suggest any gross improvement in the perfusion of the ischaemic leg.

Combined intravenous and oral pentoxifylline in the treatment of peripheral vascular disease. A clinical trial.

Sixteen patients with severe occlusive vascular disease of the lower extremities were randomised to receive a five day course of combined intravenous and oral Pentoxifylline followed by three months oral treatment only, or identical treatment with a matching placebo. Nine patients received active Pentoxifylline, and 7 placebo, Follow-up by regular clinical examination and haemoreological assessment revealed a marked improvement in claudication distance and an increase in red cell deformability in those receiving Pentoxifylline, there being no change in those receiving placebo. Although both of the above parameters were improved by the treatment, there did not appear to be a direct correlation between red cell deformability and claudication distance in individual patients. A combination of intravenous and oral Pentoxifylline therapy results in an increase in both claudication distance and red cell deformability, but the former may not te a direct consequence of the latter.

Cell seeding for small diameter ePTFE vascular grafts: a comparison between adult human endothelial and mesothelial cells.

A series of experiments was performed to compare the ability of adult human endothelial and mesothelial cells to attach and spread upon ePTFE graft material. Both cell types were harvested enzymatically and grown in culture. Two types of adhesion assay were used to assess the ability of the cultured cells to attach to ePTFE either uncoated or precoated with a variety of substrate proteins. The results showed that fibronectin, laminin, type 4 collagen and preclotted blood all greatly improve the attachment rate of endothelium to ePTFE. The extracellular matrix proteins, however, give patchy cell attachment, whereas cells seeded onto preclotted ePTFE form a virtually confluent monolayer after one hour's incubation. The matrix proteins similarly improve mesothelial attachment, although in all cases attached cells remain largely rounded up, with relatively few spreading on the surface. Mesothelial attachment to preclotted ePTFE is no better than attachment to the untreated material. It would seem that a preclotted graft seeded with endothelial cells is the combination most likely to result in a confluent monolayer within one hour.

Therapeutic retraction of the foreskin in childhood.

A retrospective study was conducted of 91 boys who had had a non-retractable but non-fibrosed prepuce treated by retraction under general anaesthesia. Of the 79 boys who had had symptoms, 67 (85%) obtained relief. Twelve of the 91 patients were later circumcised because of continuing problems. Retraction of the foreskin alone is a simple and effective alternative to circumcision in managing most boys with a symptomatic, non-retractable prepuce.

The dhnA gene of Escherichia coli encodes a class I fructose bisphosphate aldolase.

The gene encoding the Escherichia coli Class I fructose-1, 6-bisphosphate aldolase (FBP aldolase) has been cloned and the protein overproduced in high amounts. This gene sequence has previously been identified as encoding an E. coli dehydrin in the GenBanktrade mark database [gene dhnA; entry code U73760; Close and Choi (1996) Submission to GenBanktrade mark]. However, the purified protein overproduced from the dhnA gene shares all its properties with those known for the E. coli Class I FBP aldolase. The protein is an 8-10-mer with a native molecular mass of approx. 340 kDa, each subunit consisting of 349 amino acids. The Class I enzyme shows low sequence identity with other known FBP aldolases, both Class I and Class II (in the order of 20%), which may be reflected by some novel properties of this FBP aldolase. The active-site peptide has been isolated and the Schiff-base-forming lysine residue (Lys236) has been identified by a combination of site-directed mutagenesis, kinetics and electrospray-ionization MS. A second lysine residue (Lys238) has been implicated in substrate binding. The cloning of this gene and the high levels of overexpression obtained will facilitate future structure-function studies.

Effects of shear stress on endothelial cell monolayers on expanded polytetrafluoroethylene (ePTFE) grafts using preclot and fibronectin matrices.

Animal studies have shown that endothelial seeding of vascular prosthetic grafts reduces thrombogenicity and improves their patency. However, for endothelial seeding to be of clinical benefit in humans, it must withstand shear stress of blood flow. Endothelial cells labelled with Indium-111-oxine were seeded in supra-confluent densities on preclot or fibronectin coated ePTFE graft segments over a period of 90 min. These grafts with rapidly formed endothelial cell monolayers were then exposed to varying shear stresses up to a flow rate of 300 ml/min, using tissue culture medium in an artificial flow circuit. Grafts coated with preclot matrix showed 2 h cell retentions of 82.4 +/- 6.8% at 25 ml/min, 79.9 +/- 8.2% at 100 ml/min, 75.4 +/- 9.5% at 200 ml/min and 58.3 +/- 15.5% at 300 ml/min whilst those for the fibronectin matrix were 57.8 +/- 9.9%, 55.2 +/- 13.3%, 55.4 +/- 12.9% and 56.5 +/- 15.2% respectively. Overall the preclot matrix was found to be better than fibronectin (P less than 0.001). Light and scanning electron microscopy revealed well-formed endothelial cell monolayers retained on preclot matrix up to a flow rate of 200 ml/min whereas uncovered patches were seen at 300 ml/min and at all flow rates on fibronectin matrix.

Comparison of different vascular prostheses and matrices in relation to endothelial seeding.

Thin-walled expanded polytetrafluoroethylene (ePTFE), woven Dacron and gelatin-impregnated Dacron (Gelseal) vascular grafts were compared, the grafts being coated with three different matrices: collagen IV, fibronectin and preclot matrix. In addition, untreated ePTFE and Gelseal were examined. The graft segments, coated with these matrices, were incubated with radiolabelled adult human endothelial cells for 30, 60 and 90 min. Endothelial cell adherence was calculated from the ratio of radioactive counts in the grafts to counts in grafts plus supernatants. Endothelial cell attachment to untreated grafts was poor, but a suitable matrix significantly improved adherence. All three matrices tested gave good results, although preclot was best; 30-60 min incubation was sufficient for optimum cell attachment. Cell adherence to both Dacron and ePTFE was significantly better than to Gelseal. The type of prosthetic polymer and the substrate protein coating used to promote endothelial cell adherence are two important factors which may determine the ultimate success of endothelial seeding in the operating room.

Rupture of the abdominal aorta in patients with Ehlers-Danlos syndrome.

Ehlers-Danlos syndrome (EDS) is a heterogeneous inherited disorder of collagen synthesis. Type IV is frequently associated with major vascular catastrophes and challenges the vascular surgeon with its varied clinical presentation and the difficulty of vascular repair. Rupture of the abdominal aorta is one of the most serious complications and is associated with nearly 100% mortality rate. We describe here three patients with type IV EDS.

Abdominal aortic aneurysm: the role of clinical examination and opportunistic detection.

OBJECTIVES: to investigate the method of discovery of abdominal aortic aneurysms (AAA) in a district general hospital setting. DESIGN: retrospective study. MATERIALS AND METHODS: we analysed 198 patients with an AAA who presented to our unit over a 3-year period. The method of initial diagnosis, size of the AAA and whether this was palpable, irrespective of the method of detection, were recorded. RESULTS: ninety-five (48%) were discovered clinically, 74 (37.4%) during a radiological investigation, and 29 (14.6%) at laparotomy. Of the 74 AAAs first detected radiologically, subsequent physical examination showed that 28 (37.8%) were in fact palpable and missed at presentation. The average size of those discovered clinically (6. 48+/-1.32 cm) was larger than those found radiologically (5.37+/-1. 44 cm, p<0.001) or at operation (5.43+/-1.48 cm, p=0.039). The average diameter of the palpable AAAs was also greater than that of the non-palpable AAAs (6.42+/-1.24 cm vs. 4.86+/-1.38 cm, p<0.001). CONCLUSIONS: opportunistic detection of a clinically unsuspected aneurysm during clinical examination or investigation for another reason is the most common way the diagnosis of an AAA is made. Almost half of the aneurysms were diagnosed clinically, but physical examination also missed more than a third of those detected radiologically. Despite technological advancement, clinical examination still plays a paramount role in the detection of AAAs. Larger AAAs are usually palpable and more likely to be detected on clinical examination.